Impact of Propionic Acid on Liver Damage in Rats.

Propionic acid (PA) is a short chain fatty acid, a common food preservative and metabolic end product of enteric bacteria in the gut. The present study was undertaken to investigate the effect of PA on liver injury in male rats. Male western albino rats were divided into two groups. The first group served as normal control, the second was treated with PA. The activities of serum hepatospecific markers such as aspartate transaminase, alanine transaminase, and alkaline phosphatase were estimated. Antioxidant status in liver tissues was estimated by determining the level of lipid peroxidation and activities of enzymatic and non-enzymatic antioxidants. Sodium and potassium levels were also measured in liver tissue. PA treatment caused significant changes in all hepatospecific markers. Biochemical analysis of liver homogenates from PA-treated rats showed an increase in oxidative stress markers like lipid peroxidation and lactate dehydrogenase, coupled with a decrease in glutathione, vitamin C and glutathione S- transferase. However, PA exposure caused no change in sodium and potassium levels in liver tissue. Our study demonstrated that PA persuade hepatic damage in rats.

ropionic acid (PA) is a naturally occurring carboxylic acid. This dietary short-chain fatty acid occurs naturally in milk, dairy products such as yogurt and cheese (1,2). PA is mainly produced by the fermentation of indigested food by the microbiota in the colon, but can reach the blood compartment and the adipose tissue, where it reduces fatty acid levels in plasma via the inhibition of lipolysis and induction of lipogenesis in adipose tissue and suppression of fatty acid production in liver (3). PA is a fungicide and bactericide and is therefore used as a food preservative (4,5). It is also used to control fungi and bacteria growth in stored grains, hay, grain storage areas, poultry litter and drinking water (6). Pharmaceutical companies commonly include PA in the formulation of steroidal and nonsteroidal anti-inflammatory medications. Fluticasone inhalers used for respiratory conditions, antihistamine and decongestant, commonly contain PA.
PA derived from colonic bacterial fermentation contributes substantially to overall propionate load in children with disorders of propionate metabolism.The gut microbiota and its metabolites such as PA can access the brain through various routes within the liver -gut-brain axis (7,8).The these organs through the bile, hormones, inflammatory mediators, and the products of digestion and absorption. In the colon, PA is the metabolic end product of enteric bacteria, produced by fermentation of polysaccharides, oligosaccharides, long-chain fatty acids, proteins, peptides and glycoprotein precursors (9). The majority of the PA produced in the colon is absorbed, passes the colonocytes and the viscera, and drains into the portal vein. Around 90% of PA is metabolized by the liver and the rest is transported into the peripheral blood (10). Propionate affects various metabolic processes such as gluconeogenesis, ureogenesis or ketogenesis (11). However, there are also findings which appear inconsistent with the physiological role of propionate in the control of carbohydrate or lipid synthesis (12). PA is mainly metabolized in the liver and has been shown to inhibit gluconeogenis and increases glycolysis in rat hepatocytes (13). It has also been proposed that propionic acid may lower plasma cholesterol concentrations by inhibiting hepatic cholesterogenesis (14). PA influences the production of hormones through adipose tissue, such as the induction of leptin, which is a potent anorexigenic hormone and suppresses food intake through receptors expressed in the central nervous system (15).
Even though PA is necessary for normal immune and physiological functioning; elevated levels may result in disruptive effects. (16). PA can readily cross the gut-liver-blood-brain barriers and gain access to the central nervous system (17).
In the brain, it can cross cell membranes and accumulate within cells, inducing intracellular acidification (18,19) which may alter neurotransmitter releases and, ultimately, neuronal communication and behavior (20,21). In fact, propionic acidemia is a neurodevelopmental metabolic disorder characterized by elevated levels of PA that clinically resembles some aspects of autism (22).  (23). PA is neurotoxic and its axis to brain is through liver (7,8).This study attempted to investigate the possible role of neurotoxic dose of PA on liver injury in male rats as the majority of PA is metabolized by the liver.

Animals
Adult male western albino rats weighing 150-

Dosage and treatment
Rats were randomly divided into two groups with ten rats in each. The first group served as a control. On the eighth day, the animals of the second group given an oral dosage of PA at the dose of (250 mg/kg body weight/day for three days; n= ten) (24). On the third day of PA administration, the rats were scarified and the liver organ was isolated.

Sample preparation
Blood collection for estimation of AST, ALT and

Serum aspartate aminotransferase (AST)
AST was estimated by the method of Reitman and Frankel (25). The substrate, however, was 2 mM α-ketoglutarate, 0.2 M DL-aspartate, and the rest of the procedure was similar to ALT measurement method.

Serum alkaline phosphatase (ALP)
Serum alkaline phosphatase activity was measured according to the method of King and Armstrong (26), using disodium phenyl phosphate as substrate. The colour developed was read at 510 nm.

Assay of lactate dehydrogenase (LDH)
The quantitative determination of LDH in the brain homogenates was performed using the lactateto-pyruvate kinetic method described by Henry et al. (31).

Determination of potassium levels
Potassium levels were measured in a protein-free alkaline medium by reaction with sodium tetraphenyl boron, which produced a colloidal suspension. The turbidity of such a suspension is proportional to the potassium concentrations in the range of 2-7 mmol/ l (32).

Determination of sodium levels
Sodium levels were assayed by enzymatic determination of sodium, i.e., the measurement of sodium-dependent galactosidase activity using ortho-Nitrophenyl-βgalactoside (ONPG) as a substrate (33)

Statistical analysis
The values are expressed as mean ± standard error of the mean (SEM

Effect of PA on hepatic markers
The activities of AST, ALT and ALP are presented in Table 1. We observed statistically a significant increase in AST and ALP in serum of rats exposed to PA (250 mg/kg body weight/day for three days). Also the administration of PA induced a marked increase in ALT levels as compared to the control group. Table 2 presents the mean ± SEM of the GSH (µg/ml), MD (µmoles/ml) and vitamin C (µg/ml) concentrations, GST (U/ml) and LDH activities in the liver homogenates of the two groups of rats.

Effect of PA on GSH and antioxidant enzyme activities
Compared to control groups, the PA-treated rats exhibited a statistically significant increase in MD and LDH activities with a concomitant decrease of GST, GSH, and Vitamin C.

Effect of PA on sodium and potassium
In liver tissue, sodium and potassium levels were not affected by PA treatment as shown in Table 3. Figure 1 shows the percentage change of all parameters in PA treated group compared to control.

Discussion
The probably by its redox and detoxification reactions (43). The decreased concentration of GSH in liver tissue found in PA treated group of our study may be due to NADPH reduction or GSH utilization in the exclusion of peroxides (44). Support for this comes from some previous findings (21,45) in which the high levels of PA were reported to induce oxidative stress with decreased levels of total GSH in brain tissue.
Circulating antioxidants such as vitamin C are non-enzymatic scavengers of free radicals. A decrease in ascorbic acid levels in plasma (46) and liver (47) was reported in injured liver in rats.
Decreased levels of vitamin C found in PA treated rats in the present study are in line with the findings of a previous study by El-Ansary et al. (45).
Glutathione-S-transferase is actually composed of a group of isoenzymes capable of detoxifying various exogenous and endogenous substances by conjugation with glutathione. A reduction in the activity of these enzymes is associated with the accumulation of highly reactive free radicals, leading to deleterious effects such as loss of integrity and function of cell membranes (48,49).
Significant decrease of GST reported in the present study could easily be related to the oxidative effect of PA previously reported by Alfawaz et al. (50).
The active transport of sodium-potassium across the cell membrane is controlled by sodium-potassium-adenosine triphosphatase (Na+-K+-ATPase) enzyme, which is an integral plasma membrane protein responsible for a large part of the energy consumption constituting the cellular metabolic rate. Na+-K+-ATPase controls cell volume, nerve and muscle signals and drives the transport of amino acids and sugars (51). However, the non significant difference from the control value by administration of PA suggests that it may be of low toxicity to the monovalent ion.
In conclusion, increased activities of AST, ALT, and ALP suggest severe hepatic injury resulting from the administration of PA. The increase of TBARS in liver of treated rats provides evidence for the pathogenic role of PA in inducing oxidative liver injury.